Abstract: The Gut Microbiota and Graft-versus-Host Disease Hematopoietic cell transplantation (HCT) can be lifesaving for the treatment of numerous conditions, including leukemia, lymphoma, aplastic anemia, multiple myeloma, some immunodeficiencies, and other diseases. Unfortunately, graft-versus-host disease (GVHD) is a common complication of HCT, affecting up to 50% of allogeneic transplant recipients, despite human leukocyte antigen (HLA) matching between donor and recipient and despite use of immunoprophylaxis. GVHD most commonly affects the gut, liver, and skin, producing diarrhea, liver inflammation, and rashes, with varying stages (degrees) of organ involvement in different patients. GVHD is thought to arise from immune cells (e.g. T cells) in the graft that recognize host tissues as foreign and propagate an inflammatory response. It is unclear why some HCT recipients with equivalent HLA matching develop GVHD while others are spared, and why some patients with GVHD respond to immune suppression with glucocorticoid treatment while others develop steroid-refractory GVHD with high mortality. Some murine studies of bone marrow transplantation have shown that germ-free animals are protected from GHVD, and human studies have suggested that patients with GVHD have a less diverse gut microbiota early after transplant, pointing to the important role that microbes play in GVHD. This study will investigate which gut bacterial species or communities are most associated with risk of GVHD in a longitudinal study of HCT recipients where stool samples are collected weekly for 100 days, then monthly for 9 months, and subjected to broad-range 16S rRNA gene PCR with high-throughput sequencing for species-level taxonomic identification of bacteria. Stool samples from these patients will also be assayed for small molecule metabolites produced by the bacteria-host interaction that may mediate GVHD, such as short chain fatty acids and bile salts. The goal of this aim is to identify potential chemical effectors of GVHD. Metagenomic sequencing of stool DNA will be used to assess the functional and metabolic capability of gut bacterial communities to illuminate how particular bacterial communities produce metabolites linked to GVHD risk, and to identify particular bacterial species or strains with the metabolic machinery for impacting GVHD risk. Knowledge gained from this study could be used in the future to design interventions to reduce the risk of GVHD based on targeted manipulation of the gut microbiota. For example, identification of particular bacterial species or strains linked to protection from GVHD, and to the production of metabolites that mediate this protection, could lead to the development of probiotics or engineered bacterial communities for reducing GVHD risk or treating GVHD. Alternatively, enhancing the production of beneficial metabolites through exogenous administration or use of prebiotics, and blocking the production of harmful metabolites could emerge as another approach for mitigating GVHD without further immune suppression in these very compromised patients.